Circulating monocyte counts do not correlate with soluble biomarkers of inflammation and immune activation in TB-HIV co-infected individuals.

<p>(<b>A</b>) Numbers of circulating monocytes (left panel) and neutrophils (right panel) are compared at week 0 (pre-ART), at weeks 6 or at the time of IRIS, and at week 24 after ART initiation between TB-HIV co-infected patients who developed paradoxical TB-IRIS (n = 26) and those who did not (n = 22). Lines represent median values and interquartile ranges. Data were analyzed using the Mann-Whitney test or Wilcoxon matched-pairs test for paired comparisons within each study group. ** P<0.01, *** P<0.001. (<b>B</b>) The network analysis (interactome) showed statistically significant correlations (P<0.05) between neutrophil or monocyte counts and plasma biomarkers. Associations were assessed with Spearman rank tests.</p

Plasma concentrations of monocyte activation markers are increased during TB-IRIS and correlate with markers of systemic inflammation.

<p>(<b>A–C</b>) Plasma levels of soluble (s) CD14 (<b>A</b>), sCD163 (<b>B</b>) and sTF (<b>C</b>) are compared at week 0 (pre-ART), at week 6 or at the time of IRIS, and at week 24 after ART initiation between TB-HIV co-infected patients who developed paradoxical TB-IRIS (n = 26) and those who did not (n = 22). Lines represent median values and interquartile ranges. Data were analyzed using Mann-Whitney or Wilcoxon matched-pairs test for paired analyses within each study group. * P<0.05, ** P<0.01, *** P<0.001. (<b>D</b>) Left panel: A heat map was designed to depict the overall pattern of expression of plasma cytokines, chemokines and inflammatory markers in patients developing TB-IRIS vs. non-IRIS controls at week 0 (pre-ART) and at week 6 or during the time of IRIS. A two-way hierarchical cluster analysis (Ward's method) of circulating biomarkers by clinical group and time point was performed. Expression scale for each biomarker represents log₁₀ fold-change from the geometric mean of the entire study population at week 0 and week 6 or time of IRIS (n = 48 in each study time point). Right panel: The network analysis (interactome) shows statistically significant correlations (P<0.05) between all the variables measured. Data were analyzed using Spearman rank tests. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004433#ppat.1004433.s001" target="_blank">Data File S1</a> for additional details on the strength (r value) and level of significance (P-value) of each individual correlation.</p

Expression profile of plasma biomarkers of inflammation and immune activation in TB-HIV co-infected patients on anti-TB therapy.

<p>(<b>A</b>) Left panel shows <i>Mycobacterium tuberculosis</i> sputum culture grade in samples from TB-HIV co-infected individuals at study enrollment (pre-ART) stratified by time on anti-TB treatment (ATT). Data were compared using the Mann-Whitney test. Right panel shows the frequency of IRIS vs. non-IRIS patients taking ATT for different durations (≤4 weeks and>4 weeks). Data were compared using the Chi-square test. (<b>B</b>) A heat map was designed to depict the overall pattern of expression of plasma cytokines, chemokines and inflammatory biomarkers in TB-HIV co-infected patients at different time points after ATT initiation. A hierarchical cluster analysis (Ward's method) of circulating biomarkers by clinical group and time point was performed. Expression scale for each biomarker represents change from the geometric mean of the entire study population (n = 48). Differences between medians were compared using the Mann-Whitney test. * P<0.05, ** P<0.01, *** P<0.001.</p

Intracellular production of IL-6 and TNF-α by monocytes from HIV+ patients is affected by HIV plasma viremia and mycobacterial antigen load.

<p>Paired PBMC samples from eight HIV-infected individuals prior to ART initiation and after 8 weeks of treatment when patients achieved virological suppression were incubated in vitro with different doses of irradiated <i>M. tuberculosis</i> (Mtb) for 6 h in the presence of brefeldin-A. Intracellular cytokine assays for detection of IL-6 and TNF-α in monocytes (HLA-DR⁺Dump⁻ cells) were performed by multicolor flow cytometry. Gates for cytokines were set up based on fluorescent minus one controls. (<b>A</b>) Representative FACS plots of percentage of monocytes expressing IL-6 or TNF-α (left panels) following stimulation with increasing doses of Mtb. Right panel shows overlays of CD14 vs. CD16 expression on cytokine-producing cells after stimulation with irradiated Mtb 100 µg/mL and reveals that most of the cytokine-producing cells in this <i>in vitro</i> system are CD14⁺⁺CD16⁻ monocytes. (<b>B</b>) Percentage of monocytes from HIV+ patients with high or low viral loads stained positive for intracellular IL-6 after in vitro stimulation with different doses of irradiated Mtb. Data were analyzed using the Wilcoxon matched pairs test. (<b>C</b>) Percentage of monocytes from HIV+ patients with high or low viral loads stained positive for intracellular TNF-α after in vitro stimulation with different doses of irradiated Mtb. Data were analyzed using the Wilcoxon matched pairs test. * P<0.05.</p

Increased frequency of monocytes spontaneously producing pro-inflammatory cytokines pre-ART in TB-IRIS patients.

<p>Whole blood intracellular cytokine assay was performed in 17 individuals from the IRIS group and 15 non-IRIS patients from the South Indian cohort. (<b>A</b>) Gating strategy to assess intracellular cytokine production by neutrophils and monocytes/DCs and (<b>B</b>) frequency of cells producing IL-1β, IL-6 and/or TNF-α after 6 hour <i>in vitro</i> culture in the presence of brefeldin-A (unstimulated). (<b>C</b>) Pre-ART frequency of monocytes producing IL-1β, IL-6 and/or TNF-α was compared between IRIS and non-IRIS patients stratified by time on ATT or <i>M. tuberculosis</i> loads in sputum cultures. Data were analyzed using Mann-Whitney or Wilcoxon matched-pairs test for paired analyses within each study group. * P<0.05, ** P<0.01, *** P<0.001.</p

Dynamics of circulating monocyte subsets in TB-HIV co-infected patients following ART initiation.

<p>(<b>A</b>) Left panel: Representative FACS plots showing the distribution of the monocyte subsets defined by CD14 and CD16 markers at week 0 and at week 6 or time of IRIS in IRIS vs. non-IRIS patients. Monocytes were also defined according to expression of CCR2 and CX3CR1 and overlaid onto CD14 vs. CD16 graphs. Right panel: Representative FACS plots showing histograms of various phenotypic markers in monocyte subsets based on CD14 and CD16 expression by clinical group and time point. (<b>B</b>) Percentage of circulating CD14⁺⁺CD16⁻ (left), CD14⁺CD16⁺ (center) and CD14^dimCD16⁺ (right) monocytes among circulating mononuclear myeloid cells (HLADR⁺CD2⁻CD3⁻CD19⁻CD20⁻CD56⁻) were compared at week 0 (pre-ART) and at week 6 or at the time of IRIS after ART initiation between TB-HIV co-infected patients that developed paradoxical TB-IRIS (n = 26) and those who did not (n = 22). Lines represent median values and interquartile ranges. Data were analyzed using the Mann-Whitney test or Wilcoxon matched-pairs test for paired analyses within each study group. (<b>C</b>) Representative FACS plots showing co-localization of CD163 and CD14 in circulating monocytes from an IRIS patient at week 2 of ART. (<b>D</b>) The median fluorescence intensity (MFI) of CD163 expression on CD14⁺⁺CD16⁻ monocytes is compared at week 0 (pre-ART) and week 6 or at the time of IRIS after ART initiation between TB-HIV co-infected patients that developed paradoxical TB-IRIS (n = 26) and those who did not (n = 22) using the Mann-Whitney test. (<b>E</b>) Relative risk (RR) of developing paradoxical TB-IRIS per standard deviation increase in the frequency of specific monocyte subsets at week 0 (pre-ART) after log₁₀ transformation. RR were adjusted for baseline age, gender, days to ART initiation, plasma HIV RNA levels and CD4⁺ T-cell count. CI, confidence interval. * P<0.05, ** P<0.01, *** P<0.001.</p